This invention pertains to compositions and methods for selectively stimulating or inhibiting the release of follicle-stimulating hormone from the anterior lobe of the pituitary gland.
The brain controls the release of gonadotropin hormones from the anterior pituitary gland. Two important gonadotropins are follicle-stimulating hormone (FSH) and luteinizing hormone (LH). FSH is critical for spermatogenesis and for ovarian follicle development, while LH is critical to androgen secretion in males, and estrogen secretion, ovulation, and formation of the corpus luteum in females. A hormone with specific activity for releasing FSH but not LH could be used to increase fertility in humans or other animals, or to correct fertility problems caused by defective hypothalamic control of FSH secretion. Conversely, antisera or other antagonists to an FSH-specific releasing factor will inhibit FSH secretion, thereby inhibiting spermatogenesis in males, or inhibiting development of follicles and ovarian development in females, providing a new antifertility drug. It is also possible that very high doses of an FSH-specific releasing factor will inhibit FSH secretion, rather than stimulate it.
Prior indirect evidence has suggested that separate factors could be responsible for triggering the release of FSH and for triggering the release of LH in mammals. However, this hypothesis could not previously be confirmed, because no previous work successfully isolated a potent factor that selectively induces the release of FSH, but not LH. See McCann, S. et al., Control of follicle-stimulating hormone and luteinizing hormone release by hypothalamic peptides, Annals New York Academy of Sciences, 687:55-59, 1993; Dees, W. et al., Ethanol and the pulsatile release of luteinizing hormone, follicle stimulating hormone and prolactin in ovariectomized rats, Alcohol, 2:641-646, 1985; Dhariwal, A. et al., Separation of follicle-stimulating hormone-releasing factor from luteinizing hormone-releasing factor, Endocrinology, 76:290-294, 1965; Dhariwal, A. et al., Chromatographic behavior of follicle stimulating hormone-releasing factor on Sephadex and carboxy methyl cellulose, Neuroendocrinology, 2:294-303, 1967; Igarashi, M. et al., A hypothalamic follicle stimulating hormone-releasing factor, Endocrinology, 74:446-452, 1964; Lumpkin, M. et al., Effect of destruction of the dorsal anterior hypothalamus on follicle-stimulating hormone secretion in the rat, Endocrinology, 115:2473-2480, 1984; Samson, W. et al., Chromatographic and biologic analysis of ME and OVLT LHRH, Peptides, 1:97-102, 1980; Mizunuma, H., et al., Evidence for an FSH-releasing factor in the posterior portion of the rat median eminence, Life Sci., 33:2003-2009, 1983.
Luteinizing hormone releasing hormone (LHRH, also known as gonadotropin-releasing hormone, or GnRH) has both LH-releasing activity and FSH-releasing activity. See Schally, A. et al., Gonadotropin-releasing hormone: one polypeptide regulates secretion of luteinizing and follicle-stimulating hormones, Science, 173:1036-1038, 1971; and D. Lincoln, Gonadotropin-releasing hormone (GnRH): basic physiology, pp. 218-229 in L. DeGroot et al., Endocrinology, 1995. The latter states at page 218: xe2x80x9cThere is no convincing evidence for the existence of a separate and specific FSH-releasing hormone, although some components of the GnRH precursor and some GnRH analogues appear to differ in the degree to which they stimulate the secretion of the two gonadotropins.xe2x80x9d
Sower, S. et al., Primary structure and biological activity of a third gonadotropin-releasing hormone from lamprey brain, Endocrinology, 132:1125-1131, 1993 reported the structure of lamprey GnRH-III (referred to as l-LHRH-III in this specification), and reported that it stimulated estradiol and progesterone release from Petromryzon marinus (lamprey) ovaries. (Lampreys, jawless fish, are representatives of what is generally considered to be the most primitive of the extant classes of vertebrates.)
Lamprey l-LHRH-I has been reported to have relatively low activity in releasing either FSH or LH in rats. Yu, W. et al., Selective FSH-releasing activity of [D-Trp9]GAP1-13: comparison with gonadotropin-releasing abilities of analogs of GAP and natural LHRHs, Brain Res. Bull., 25:867-873, 1990.
Schally, A. et al., Re-examination of porcine and bovine hypothalamic fractions for additional luteinizing hormone and follicle stimulating hormone-releasing activities, Endocrinology, 98:380-391, 1976 reported that in vivo FSH-releasing activity could not be separated from LH-releasing activity from porcine hypothalami by fractionation on Sephadex, and concluded that there was only one gonadotropin-releasing hormone (GnRH).
By contrast, Lumpkin, M. et al., Purification of FSH-releasing factor: Its dissimilarity from LHRH of mammalian, avian, and piscian origin, Brain Res. Bull., 18:175-178, 1987 reported that FSH-releasing activity was separated from the LH-releasing activity in ovine hypothalami on Sephadex G-25, but did not isolate the factor causing FSH release.
Neurons that are immunopositive for l-LHRH-I have been identified in human hypothalami, projecting from the arcuate region to the median eminence. Stopa, E. et al, Polygenic expression of gonadotropin-releasing hormone (GnRH) in human?, Peptides, 9:419-423, 1988.
Lincoln, D., Luteinizing Hormone-Releasing Hormone, pp. 142-151 in DeGroot et al. (ed), Endocrinology, 1989, discloses various agonists and antagonists for mammalian LHRH.
W. Yu et al., xe2x80x9cA hypothalamic follicle-stimulating hormone-releasing decapeptide in the rat,xe2x80x9d Proc. Natl. Acad. Sci. USA, 94:9499-9503, 1997 discloses some of the work reported in the present specification, but is not believed to constitute prior art.
U.S. Pat. No. 4,973,577 discloses a 28,000 dalton protein isolated from porcine follicular fluid that stimulates the release of FSH, but not of LH. This protein has a relatively slow onset of action, and is relatively difficult to synthesize. The protein was said to be a homodimer of two chains of 116 amino acid residues each, or 232 residues total.
U.S. Pat. No. 3,888,836 discloses a method for synthesizing mammalian LHRH. Mammalian LHRH causes increased serum levels of both LH and FSH.
U.S. Pat. No. 4,721,775 discloses certain peptides that non-selectively induce the secretion of both LH and FSH.
Attempts in our laboratory to purify FSH-releasing factor (FSH-RF) by fractionation of lamb hypothalami (discussed in some of the papers cited above) were successful only at certain seasons of the year, and even then we found that activity was lost after samples were stored at xe2x88x9220xc2x0 C. (unpublished data). Thus our prior work did not successfully isolate or identify the putative FSH-releasing factor.
Other studies in our laboratory confirmed FSH-releasing activity by incubating stalk-median eminence (SME)-extracts in vitro with hemipituitaries from male rats. We confirmed the FSH-releasing activity of sheep and rat SME extracts in this assay, and found that the FSH-releasing activity emerged from columns of Sephadex G-25 just prior to emergence of LHRH, similar to results we had seen in an in vivo assay in ovariectomized, estrogen- and progesterone-blocked female rats. Even where we were able to extract crude or partially purified fractions showing selective FSH-releasing activity, that activity was relatively low compared to the activity of fractions with LH-releasing activity (unpublished data).
Our laboratory also screened known LHRH""s from various species for selective FSH-releasing activity; and we also evaluated the activity of 25 analogs of LHRH in in vivo assays. (LHRH""s from various species are disclosed in Lumpkin et al., 1987.) One analog was found to have only FSH-releasing activity, but its potency was very low, and the slope of its dose-response curve was flat. Of the known forms of LHRH from other species, we found that only chicken (c) LHRH-II had slightly preferential FSH-releasing activity in vivo (unpublished data).
We have unexpectedly discovered that l-LHRH-III is the long-sought FSH-releasing factor. This activity has been confirmed in vitro by incubation with hemi-anterior pituitaries of adult male rats. Following intravenous injection at the lowest dose tested to date (10 picomoles), this peptide produced an increase in FSH in vivo (P less than 0.01) within ten minutes, but no significant increase in LH. Such a selective effect has not previously been reported for any analog of mammalian LHRH.
General
Adult male and female Sprague-Dawley rats (Holtzmann, Madison, Wis.; 200-250 g) were housed two per cage under controlled conditions of temperature (23-25xc2x0 C.) and lighting (on from 0500 to 1700 hr). The animals had free access to a pellet diet and to tap water.
The l-LHRH-III used in the experiments reported here was synthesized by standard solid-state peptide synthesis methods, and was purified to greater than 97% purity by preparative reverse-phase high performance liquid chromatography. All other peptides used were purchased from Peninsula Laboratories (Belmont, Calif.), except as otherwise noted.
The significance of differences among multiple groups was determined by analysis of variance, with subsequent Newman-Keuls multiple comparisons at each point. Student""s t-test was used to determine the significance of differences between two groups.
In Vitro Studies
After acclimatization for 5 or more days in the vivarium, male rats were killed by decapitation. Following removal of the posterior lobe, the anterior pituitary (AP) was bisected longitudinally, and each AP was incubated in a tube containing 0.5 ml Krebs-Ringer bicarbonate (5 mM ascorbic acid; pH 7.4) buffer (KRB) in an atmosphere of 95% O2/5% CO2 in a Dubnoff shaker (50 cycles per min) for a period of 60 min. Following this pre-incubation period, the APs were incubated for 3 hr in fresh KRB buffer alone (control), or in KRB containing one of various concentrations of synthetic mammalian (m)-LHRH, l-LHRH-III, l-LHRH-I, chicken (c)-LHRH-II, or salmon (s)-LHRH. The medium was then aspirated, and was stored frozen at xe2x88x9220xc2x0 C. until radio-immunoassays (RIA) for FSH and LH were conducted. FSH and LH were measured using kits supplied by the National Institute of Arthritis Digestive Diabetes and Kidney Disease; hormone values were expressed in terms of NIH-rFSH-RP-2 and NIH-rLH-RP-3 standards. The experiments were repeated twice. The inter- and intra-assay coefficients of variation for FSH assays were 7.5% and 5.2%, respectively; and were 6.5% and 4.8% for LH assays.
In Vivo Studies
Female rats were ovariectomized under anesthesia with isoflurane 4 weeks prior to the in vivo experiments. Three days before blood sampling, each ovariectomized rat was injected subcutaneously with 50 xcexcg estradiol benzoate (Sigma Chemical Co., St. Louis, Mo.) dissolved in 0.1 ml sesame oil, and 25 mg progesterone (Eli Lilly, Indianapolis, Ind.) dissolved in 0.5 ml sesame oil. One day prior to testing, each animal was implanted with a jugular-atrial catheter. On the morning of testing, polyethylene tubing (PE-50) was connected to the distal end of the jugular-atrial catheter on the rat""s dorsum to facilitate blood sampling and intravenous injection. Animals were acclimatized one hour before initial blood samples were taken. Heparinized blood samples (5 ml) were collected just before, and at 10, 30, and 60 minutes after injection of 0.5 ml isotonic saline or l-LHRH-III (10 or 100 pmole) in 0.5 ml isotonic saline. After removal of each blood sample, an equal volume of isotonic saline was administered to maintain blood volume.
Effects of l-LHIRH-I, l-LHRH-III, and Mammalian (m)-LHRH on FSH Release In Vitro
Lamprey l-LHRH-III caused FSH release in a dose-related fashion, with a minimal effective concentration (MEC) of 10xe2x88x929 M (the lowest concentration tested) or lower, and a maximal effect at about 10xe2x88x926 M. Thereafter, release of FSH levelled off through the highest concentration tested (10xe2x88x924 M).
Lamprey l-LHRH-I caused a small but statistically significant release of FSH at a concentration of 10xe2x88x925 M; the amount of FSH released was significantly less than that caused by l-LHRH-III at the two concentrations tested (10xe2x88x926 and 10xe2x88x925 M). By contrast, m-LHRH produced equivalent levels of FSH release at the two concentrations tested (4xc3x9710xe2x88x929 and 2xc3x9710xe2x88x928 M) as compared to the levels induced by l-LHRH-III. See Table 2.
Effect of l-LHRH-I, l-LHRH-III, and m-LHRH on LB Release In Vitro
In contrast to its effects on FSH release, l-LHRH-III had a much weaker effect on LH release. In fact, l-LHRH-III only caused the release of significant and comparable concentrations of LH at the three highest concentrations tested (10xe2x88x926-10xe2x88x924 M). We found that l-LHRH-I was inactive at the two concentrations tested (10xe2x88x926 and 10xe2x88x925 M). Mammalian LHRH gave a dose-related stimulatory effect on LH release, and was active at the lowest concentration tested (4xc3x9710xe2x88x929 M). See Table 3.
Effects of Salmon and Chicken LHRH-II on Gonadotropin Release In Vitro
Salmon (s)-LHRH stimulated the release of both FSH and LH at the two concentrations tested (10xe2x88x927 and 10xe2x88x926 M). See Tables 4 and 5. Chicken (c) LHRH-II stimulated LH release at doses from 10xe2x88x928 to 10xe2x88x926 M, but the dose effect was not statistically significant. The c-LHRH-II had equivalent LH-releasing activity to that of m-LHRH, but l-LHRH-III showed no LH-releasing activity in this experiment. The c-LHRH-II only significantly increased FSH release at the highest concentration tested (10xe2x88x926 M). In this experiment, the MEC for l-LHRH-III for a statistically significant release of FSH was two orders of magnitude lower than that for c-LHRH-II.
Effects of l-LHRH-III on FSH and LH Release In Vivo
Saline-injected control animals experienced a significant decline in plasma FSH levels 10 minutes after injection, followed by a return to levels that did not differ significantly from pre-injection FSH levels by 30- and 60-minutes post-injection (data not shown).
Compared to the control animals, animals injected with the lowest dose of l-LHRH-III (10 pmole) showed a highly significant increase in plasma FSH 10 minutes after injection, an effect that vanished by 30 minutes post-injection. See Table 6. Increasing the dose of l-LHRH-III to 100 pmole produced a slightly greater effect at 10 minutes that was maintained 30 minutes after injection. Thus the 100 pmole dose caused a more prolonged effect than the 10 pmole dose.
Injection of saline diluent produced a slight decrease in LH plasma concentration 10 minutes post-injection, an effect that continued for the duration of the experiment, but that was not statistically significant. Neither the 10 pmole nor the 100 pmole dose of l-LHRH-III produced a statistically significant difference in LH levels versus saline control at any of the times measured. See Table 7.
Thus l-LHRH-III caused the release of FSH in vivo, but not LH, at each of the two doses tested, 10 pmole and 100 pmole.
As these results have demonstrated, l-LHRH-III is the first highly specific and potent FSH-releasing peptide discovered. The l-LHRH-III behaves completely differently from the other polypeptides tested. We found that l-LHRH-I had minimal potency to release either FSH or LH. Salmon LHRH and chicken LHRH-II had low potency, and lacked specificity for FSH release. The in vivo assays using the other known vertebrate LHRH""s showed that only one of them, c-LHRH-II, possessed even slight selectivity for FSH release.
We conclude that l-LHRH-III is a highly conserved peptide in vertebrates, and in particular that it or a closely related peptide is the mammalian peptide hormone responsible for the potent and specific release of FSH. If l-LHRH-III is not identical to the mammalian FSH-RF, the degree of homology between the two is quite high.
Mammalian m-LHRH and l-LHRH-III were approximately equipotent toward releasing FSH. The decreased potency of l-LHRH-III toward releasing LH is probably accounted for by the fact that l-LHRH-III only has 60% homology with m-LHRH (see Table 1). The differences in sequences are accounted for by the differing amino acids in positions 5-8, which presumably cause a drastic decrease in LH-releasing activity and an increase in FSH-releasing capabilities. It is probable that the tetrapeptide l-LHRH-III 5-8 binds to the active site of a putative specific FSH-RF receptor. Presumably, the FSH-RF receptor confers this specificity for FSH release, whereas the LHRH receptors stimulate the release of both hormones, albeit with a greater sensitivity for LH than FSH release. FSH-RF receptors may reside on gonadotropes that contain only FSH. LHRH receptors may cause release of both hormones from gonadotropes that contain both FSH and LH. LHRH receptors may also be located on gonadotropes that only contain LH.
Pulsatile gonadotropin release in the rat is characterized by simultaneous pulses of FSH and LH, by pulses of LH alone, and by pulses of FSH alone. We hypothesize that the first two types of pulses may be accounted for by LHRH, and the third by FSH-RF.
The discovery of FSH-RF has important implications for veterinary and human medicine. For example, treatment of farm animals with FSH-RF should lead to maturation of increased numbers of ovarian follicles and subsequent ovulations, leading to increased litter sizes. FSH-RF may be used as a drug to increase fertility in humans.